Outline

  • Abstract
  • Keywords
  • 1. Introduction
  • 2. Materials and Methods
  • 2.1. Preparation of Bovine Monocyte-Derived Macrophages (bmdm)
  • 2.2. Sirna Duplexes
  • 2.3. Transfection
  • 2.4. Electroporation
  • 2.5. Quantification of Sirna Uptake and Cytotoxicity
  • 2.6. Quantitative (q)rt-Pcr Analysis of Target Gene Knock-Down and Interferon Response
  • 3. Results and Discussion
  • 3.1. Delivery of Sirna by Transfection Reagents
  • 3.1.1. Screening of Transfection Reagents
  • 3.1.2. Investigation of Potential Off-Target Effects of Transfection Reagents
  • 3.1.3. Target Gene Knock-Down Using Transfection Reagents
  • 3.2. Delivery of Sirna by Electroporation
  • 3.2.1. Optimization of Sirna Uptake by Electroporation
  • 3.2.2. Investigation of Target Gene Knock-Down and Potential Off-Target Effects with Electroporation
  • 3.3. Conclusions
  • Conflict of Interest Statement
  • Acknowledgements
  • Appendix A. Supplementary Data
  • References

رئوس مطالب

  • چکیده
  • کلید واژه ها
  • 1.مقدمه
  • 2. مواد و روش
  • 2.1. آماده سازی ماکروفاژهای مشتق شده از مونوسیت گاوی (bMDM)
  • 2.2. siRNA دورشته ای
  • 2.3. ترانسفکشن
  • 2.4. الکتروپوریشن
  • 2.5. تعیین کمیت جذب و سمیت سلولی siRNA
  • 2.6. تجزیه و تحلیل RT-PCR کمی از کار افتادن ژن هدف و پاسخ اینترفرون
  • 3. نتایج و بحث
  • 3.1. تحویل siRNA توسط معرف های ترانسفکشن
  • 3.1.1. غربالگری معرف های ترانسفکشن
  • 3.1.2. بررسی اثرات بالقوه off-target معرف های ترانسفکشن
  • 3.1.3. از کار انداختن ژن هدف با استفاده از معرف های ترانسفکشن
  • 3.2. تحویل siRNA توسط الکتروپوراسیون
  • 3.2.1. بهینه سازی جذب انتقال siRNA توسط الکتروپوراسیون
  • 3.2.2. بررسی از کار افتادن ژن هدف و اثرات بالقوه هدف خاموش با الکتروپوراسیون
  • 3.3. نتیجه گیری

Abstract

The manipulation of the RNA interference pathway using small interfering RNA (siRNA) has become the most frequently used gene silencing method. However, siRNA delivery into primary cells, especially primary macrophages, is often considered challenging. Here we report the investigation of the suitability of two methodologies: transient transfection and electroporation, to deliver siRNA targeted against the putative immunomodulatory gene Mediterranean fever (MEFV) into primary bovine monocyte-derived macrophages (bMDM). Eleven commercial transfection reagents were investigated with variable results with respect to siRNA uptake, target gene knock-down, cell toxicity and type I interferon (IFN) response induction. Three transfection reagents: Lipofectamine 2000, Lipofectamine RNAiMAX and DharmaFECT 3, were found to consistently give the best results. However, all the transfection reagents tested induced an IFN response in the absence of siRNA, which could be minimized by reducing the transfection reagent incubation period. In addition, optimized siRNA delivery into bMDM by electroporation achieved comparable levels of target gene knock-down as transient transfection, without a detectable IFN response, but with higher levels of cell toxicity. The optimized transient transfection and electroporation methodologies may provide a starting point for optimizing siRNA delivery into macrophages derived from other species or other cells considered difficult to investigate with siRNA.

Keywords: - - - -

Conclusions

The results reported here demonstrate that, contrary to the dogma that primary macrophages are difficult to transfect, primary bMDM can be electroporated or transfected with siRNA resulting in good levels of target gene silencing. The methodologies described above have now been used to successfully silence nine genes in bMDM (manuscripts in preparation). It is hoped that these methodologies will provide a starting point for optimizing siRNA use in primary macrophages from other species and other primary cells which are regarded as being hard to transfect, e.g. dendritic cells. Several of the tested transfection reagents appear suitable for use: DharmaFECT 3, Lipofectamine 2000 and RNAiMAX. Electroporated siRNA silenced the MEFV gene to a comparable level as transfected siRNA, but the procedure resulted in more cell death and the remaining cells were less robust than transfected cells in down-stream activation studies (data not shown). The transfection protocols allow siRNA uptake by adhered cells and therefore it is much easier to reapply siRNA to bMDM, thereby extending the period of gene silencing, than by electroporation. The choice of transfecting or electroporation siRNA into cells depends on the individual experiments. The fragility of electroporated cells to challenge means that the use of transfection reagents is more suitable than electroporation for our work investigating the role of host macrophage genes in the response to infection. However, the increased expression of MEFV with transfection reagent treatment illustrates that both methodologies do affect macrophages and highlights the importance of the inclusion of suitable controls in siRNA experiments.

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